Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.

The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Author Correction: EPO does not promote interaction between the erythropoietin and beta-common receptors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

EPO does not promote interaction between the erythropoietin and beta-common receptors.

A direct interaction between the erythropoietin (EPOR) and the beta-common (ßc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and ßc receptor in tissue protection while others have shown no involvement of the ßc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the ßc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and ßc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the ßc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the ßc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the ßc receptor do not directly interact and that the IRR is not involved in anaemic stress.

The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling.

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.

Role of the ß Common (ßc) Family of Cytokines in Health and Disease.

The ß common ([ßc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use ßc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the ßc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the ßc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease.

Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

The GM-CSF, IL-3, and IL-5 receptors constitute the ßc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while ßc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal.

CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common ß chain of the IL-3, GM-CSF and IL-5 receptors.

The ß common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared ß common (ßc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human ßc receptor. The binding epitope of CSL311 on the ßc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human ßc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 ß common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human ßc receptor is central to pathogenesis. The coordinates for the ßc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).

The ßc receptor family - Structural insights and their functional implications.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (ßc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure-function studies, giving new insights into cytokine-receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the ßc receptor family are being used to discover novel and disease-specific therapeutics.

Dual mechanism of interleukin-3 receptor blockade by an anti-cancer antibody.

Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas "open-like" IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a "double hit" cytokine receptor blockade.

Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.

Signalling by the ßc family of cytokines.

The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the ßc family due to their receptors sharing the signalling subunit ßc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the ßc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the ßc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.

The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM-CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure-function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM-CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.

Activation of aldo-keto reductase family member 1B14 (AKR1B14) by bile acids: Activation mechanism and bile acid-binding site.

Aldo-keto reductase (AKR) 1B14, a rat ortholog of mouse androgen-dependent vas deferens protein (AKR1B7), is involved in the synthesis of prostaglandin F(2α) and detoxification of 4-oxononenal formed by lipid peroxidation. The NADPH-linked reductase activity of AKR1B14 was activated by various bile acids. Although the activation was increased by decreasing pH from 9.0 to 6.0, the concentrations giving maximum stimulation (2- to 18-fold) were 0.2-6.0 µM for bile acids at pH 7.4. Kinetic analyses of the activation by glycochenodeoxycholic acid in the forward and reverse reactions, together with fluorescence changes and protection against 4-oxononenal-induced inactivation by bile acid, indicate that the bile acid binds to the enzyme and its coenzyme binary complex as a non-essential activator. The bile acid binding to AKR1B14 mainly accelerates the NADP(+) dissociation, the rate-limited step of the enzyme reaction. AKR1B7 was also activated by bile acids, but the activation was low and independent of pH. The mutagenesis of His269 and Leu267 of AKR1B14 into the corresponding residues (Arg and Pro, respectively) of AKR1B7 resulted in low and pH-independent activation by bile acids. The results, together with the docking of the bile acid in the recently determined crystal structure of AKR1B14, identify the bile acid-binding site of which His269 plays a key role in significant activation through its electrostatic interaction with the carboxyl group of bile acid, facilitating the release of NADP(+).

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis.

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K(i)=0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K(i) value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K(i) values compared to the wild-type enzyme, while the remaining mutations had little or no effects.

Structure of rat aldose reductase-like protein AKR1B14 holoenzyme: Probing the role of His269 in coenzyme binding by site-directed mutagenesis.

Rat aldose reductase-like protein (AKR1B14) is the ortholog of mouse vas deferens protein (AKR1B7) playing roles in detoxification of reactive aldehydes and synthesis of prostaglandin F(2α). The crystal structure of the binary complex (AKR1B14-NADPH) was determined at 1.86Å resolution, and showed that the adenine ring and the 2'-phosphate group of the coenzyme formed π-stacking and electrostatic interactions with the imidazole ring and ND1 atom, respectively, of His269, which is not conserved in other aldose reductase-like proteins. The interactions were supported by site-directed mutagenesis of His269 to Arg, Phe and Met, which increased the K(m) for NADPH by 4, 7 and 127-fold, respectively. This is the first report of the tertiary structure of a rodent AKR1B7 ortholog, which describes the role of a novel dual interaction for the non-conserved His269 in coenzyme binding.

Inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1).

Human 20α-hydroxysteroid dehydrogenase (AKR1C1), a member of the aldo-keto reductase (AKR) superfamily, is one of four isoforms (with >84% amino acid sequence identity) existing in human tissues. AKR1C1 most efficiently reduces biologically active progesterone and 5α-pregnan-3α-ol-20-one into their corresponding 20α-hydroxysteroids among the isoforms. The enzyme also accepts endogenous and xenobiotic non-steroidal carbonyl compounds as the substrates. In addition to the up-regulation of the AKR1C1 gene in cancer cells, the enzyme's over-expression in the cells of lung, ovary, uterine cervix, skin and colon carcinomas was reported to be associated with resistance against several anticancer agents. Thus, AKR1C1 may be a marker of the above cancers and a target of poor prognosis in cancer therapy. The recently determined X-ray crystal structures of AKR1C1/NADP(+)/20α-hydroxyprogesterone and AKR1C1/NADP(+)/3,5-dichlorosalicylic acid ternary complexes have provided a strong foundation for structure-based design methods to improve inhibitor selectivity and potency. In this review we provide an overview of the different types of AKR1C1 inhibitors and an update on the design of potent and selective inhibitors based on the crystal structure of the enzyme-inhibitor complex. Article from the Special issue on Targeted Inhibitors.

Structure-based optimization and biological evaluation of human 20α-hydroxysteroid dehydrogenase (AKR1C1) salicylic acid-based inhibitors.

The tertiary structure of the Leu308Val mutant of human 20α-hydroxysteroid dehydrogenase (AKR1C1) in complex with the inhibitor 3,5-dichlorosalicylic acid (DCL) has been determined. Structures and kinetic properties of the wild-type and mutant enzymes indicate that Leu308 is a selectivity determinant for inhibitor binding. The Leu308Val mutation resulted in 13-fold and 3-fold reductions in the inhibitory potencies of DCL and 3-bromo-5-phenylsalicylic acid (BPSA), respectively. The replacement of Leu308 with an alanine resulted in 473-fold and 27-fold reductions in the potencies for DCL and BPSA, respectively. In our attempts to optimize inhibitor potency and selectivity we synthesized 5-substituted 3-chlorosalicylic acid derivatives, of which the most potent compound, 3-chloro-5-phenylsalicylic acid (K(i) = 0.86 nM), was 24-fold more selective for AKR1C1 relative to the structurally similar 3α-hydroxysteroid dehydrogenase (AKR1C2). Furthermore, the compound inhibited the metabolism of progesterone in AKR1C1-overexpressed cells with an IC(50) value equal to 100 nM.

Factorizing the role of a critical leucine residue in the binding of substrate to human 20alpha-hydroxysteroid dehydrogenase (AKR1C1): molecular modeling and kinetic studies of the Leu308Val mutant enzyme.

A comparison of the structures and kinetic properties of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1) and its mutant enzymes (Leu308Val and Leu308Ala) indicates that Leu308 is a selectivity determinant for substrate binding. While the Leu308Val mutation improved the catalytic efficiency (k(cat)/K(m)) of AKR1C1 towards the two substrates 5alpha-pregnane-3alpha,20alpha-diol (PregA) and 5beta-pregnan-3alpha-ol-20-one (PregB), the Leu308Ala mutation rendered the enzyme inactive. In the docked model of PregA the conformation of the steroid molecule was similar to that of 20alpha-hydroxyprogesterone in the crystal structure of the AKR1C1 complex where the steroid did not interact with the catalytic residues Tyr55 and His117. In the case of PregB the steroid interacted with the catalytic residue His117 and formed close contacts with Leu308, suggesting that the binding mechanism of 3alpha-hydroxysteroids in the active site of AKR1C1 is different from that of 20alpha-hydroxysteroids.